Effect of the Strawberry Genotype, Cultivation and Processing on the Fra a 1 Allergen Content

Birch pollen allergic patients show cross-reactivity to vegetables and fruits, including strawberries (Fragaria × ananassa). The objective of this study was to quantify the level of the Fra a 1 protein, a Bet v 1-homologous protein in strawberry fruits by a newly developed ELISA, and determine the effect of genotype, cultivation and food processing on the allergen amount. An indirect competitive ELISA using a specific polyclonal anti-Fra a 1.02 antibody was established and revealed high variability in Fra a 1 levels within 20 different genotypes ranging from 0.67 to 3.97 μg/g fresh weight. Mature fruits of red-, white- and yellow-fruited strawberry cultivars showed similar Fra a 1 concentrations. Compared to fresh strawberries, oven and solar-dried fruits contained slightly lower levels due to thermal treatment during processing. SDS-PAGE and Western blot analysis demonstrated degradation of recombinant Fra a 1.02 after prolonged (>10 min) thermal treatment at 99 ◦ C. In conclusion, the genotype strongly determined the Fra a 1 quantity in strawberries and the color of the mature fruits does not relate to the amount of the PR10-protein. Cultivation conditions (organic and conventional farming) do not affect the Fra a 1 level, and seasonal effects were minor

Effect of tomato variety, cultivation, climate and processing on Sola l 4, an allergen from Solanum lycopersicum

Tomatoes (Solanum lycopersicum) are one of the most consumed vegetables worldwide. However, tomato allergies in patients suffering from birch pollen allergy occur frequently. Due to highly similar protein structures of the tomato allergen Sola l 4 and the major birch pollen allergen Bet v 1, patients cross-react with allergenic proteins from tomato as well as other fruits or vegetables. The aim of this study was to quantify Sola l 4 in various tomatoes differing in color, size and shape for identification of varieties with a reduced allergen level. Therefore, an indirect competitive ELISA using a specific polyclonal Sola l 4 antibody was developed. In addition, two varieties, both cultivated either conventionally or organically and furthermore dried with different methods, were analyzed to investigate the influence of the cultivation method and processing techniques on Sola l 4 level. Within 23 varieties, Sola l 4 content varied significantly between 0.24 and 1.71 μg Sola l 4/g FW. The tomato cultivars Rugantino and Rhianna showed the significantly lowest level, whereas in cultivars Farbini and Bambello the significantly highest concentration was determined. Drying of tomatoes in the oven and by sun resulted in a significant decrease. The thermal instability was verified for the recombinant Sola l 4 emphasizing the results for the native protein in dried tomato samples. Overall, the Sola l 4 content is cultivar-dependent and no correlation between color and Sola l 4 amount was found. During the drying process of tomatoes Sola l 4 level was significantly reduced due to thermal instability. Growing conditions have a minor effect whereas seasonal effects show a more pronounced impact. These findings could extend the knowledge about the allergen level of different tomato varieties and may help to improve food safety to potentially increase the life quality of patients suffering from birch pollen allergy

Development of displacement electrochemical inmunosensors: the case of 2,4,6-trichloroanisole

Development of displacement El objetivo de este trabajo es explorar y explotar los principios de funcionamiento de un Inmunosensor Electroquímico de Desplazamiento [(Displacement Electrochemical Immunosensor (DEI)] y también del ELISA Indirecto Competitivo [(Indirect Competitive ELISA (ICE)] para la detección de 2,4,6-tricloroanisol (TCA). Para tal fin, se lleva a cabo el desarrollo racional de un ELISA Indirecto Competitivo. El ensayo desarrollado resulta capaz de detectar TCA en concentraciones 1ppt a 1 ppm, con un limite de detección de 4.2 ppt. El ensayo desarrollado puede tener un particular interés comercial en situaciones donde el tiempo experimental requerido es de menos de 80 minutos.Se desarrolla también un modelo matemático (MM) cuyo principal objetivo es permitir el desarrollo racional de un Inmunosensor Electroquímico de Desplazamiento (DEI). A pesar de las bajas constantes de afinidad observadas en los anticuerpos obtenidos para este trabajo, se logra desarrollar un DEI funcional cuyo limite de detección de TCA (0.2 ppm) se corresponde con los valores obtenidos a través del MM.La adsorción inespecífica (NSA) de proteínas es identificada como uno de los problemas críticos que impidieron alcanzar limites de detección más bajos. El uso del electroquímicamente compatible Cu UPD como barrera/control de la NSA, junto con la detección amperométrica del desplazamiento son propuestos en este trabajo como base o punto de partida para el desarrollo de inmunosensores que puedan ser operados sin necesidad de marcaje (labelling) o la adición de otros componentes diferentes de la muestra de interés (reagentless and labelless immunosensors).The purpose of this work is to explore and exploit the principles of Displacement Electrochemical Immunosensing (DEI) and Indirect Competitive ELISA (ICE) to detect 2,4,6-trichloroanisole (TCA). The rational design of indirect competitive ELISA for TCA detection is attempted. The developed assay detects TCA at concentrations from 1ppt to 1 ppm, with a limit of detection of 4.2 ppt. The assay can be commercially useful in situations where less than 80 minutes total assay time is required. A mathematical model (MM) is developed for the rational design of an electrochemical displacement immunosensor (DEI). Despite the low affinity constants of the antibodies obtained for this work a functional DEI is developed with the predicted by the MM high limit of detection for TCA (0.2 ppm). The non-specific adsorption (NSA) of proteins is identified as a critical problem inhibiting further optimization of the DEI. The use non-insulating Cu UPD as NSA controller or electrochemically compatible blocking, together with amperometric displacement detection are proposed as a platform that could permit further development of reagentless and labelless immunosensors

Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine.

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules

Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

AbstractAflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples

Amperometric immunoassay of azinphos-methyl in water and honeybees based on indirect competitive ELISA

An electrochemical immunosensor based on indirect competitive ELISA technique has been developed and tested for the detection of azinphos-methyl in aqueous solutions and spiked honeybee extracts. The detection of the pesticide was based on competition for binding to monoclonal antibodies with an ovalbumin (OVA) conjugate, followed by the incubation with anti-mouse IgG labeled with horseradish peroxidase, whose activity was measured amperometrically with hydroquinone as the substrate. The sensitivity of the azinphos-methyl assay, estimated as the IC50 value, was found to be 1.2 nmol L-1 (60 min incubation), with a linear range of 0.6-500 nmol L-1 in optimal conditions. The matrix effect on the detection of azinphos-methyl in honeybee extract was found negligible, with the recovery values in the range 92-105%. Copyright © Taylor & Francis Group, LLC

Occurrence of Fungi of Public Importance in Rodents Trapped along and inside Grain Storage Facilities in Mbeya Municipal, Tanzania.

This work was supported by the African Centre of Excellence for Innovative Rodent Pest Management and Biosensor Technology Development (ACE IRPM and BTD) at the Institute of Pest Management of the Sokoine University of Agriculture.   Abstract Rodents act as agents for the dispersal of pathogenic entities including fungi and enable their colonization in new areas. They interact with the human environment which acts as a route for the transmission of pathogens. A total of 210 rats were trapped in and along the storage facilities in selected wards in Mbeya city. Fresh fecal samples were collected from the intestines by dissecting the abdominal part of the rodent to obtain pellets. Samples were kept in clean envelopes and preserved at -20 0C at Mbeya National Research Institute (NIMR) for further laboratory analysis. Fungi were isolated by culturing in selective media and identification was done by colony morphology. Further confirmation of the isolated Aspergillus flavus was done by nested PCR to confirm the presence flR the gene in the isolates. Aflatoxigenicity of the isolated A. flavus was tested with a controlled experiment in which non-contaminated maize kernels were inoculated with the fungal spores and incubated for up to 15 days and accumulation of the aflatoxin analyzed by indirect competitive ELISA. Aspergillus fumigatus was the dominant fungal specie from the cultured samples, with a prevalence of 26% followed by Aspergillus niger and Fusarium species, both with a prevalence of 9%, Aspergillus flavus 3% and Aspergillus ochraceus 1%.  Indirect competitive ELISA was performed on 10 maize samples that were infected with A. flavus isolates, 10 maize samples free from isolates contamination, and 4 pure isolates of A. flavus to check whether the isolates were potential producers of aflatoxins. The four pure isolates had a high concentration of aflatoxin compared to the samples contaminated with A. flavus isolates. These findings justify that rodents harbor pathogenic fungi in their intestinal tracts and act as dispersal agents of the fungi to foods and other human and animal premises. Effective control measures should therefore be applied in protecting foods and premises from rodents, especially mice and rats to minimize risks of disease spread. Keywords: Pathogenic fungi, Rodents, Aspergillosis, dispersal, and mycotoxin-producing fungi. DOI: 10.7176/JHMN/109-05 Publication date:July 30th 202

Determination of lactadherin concentration in dairy by-products by ELISA: Effect of heat treatment and hydrolysis

Lactadherin is a peripheral glycoprotein of the milk fat globule membrane with several attributed biological activities. In this study, we developed an indirect competitive ELISA to determine lactadherin concentration by using a rabbit polyclonal antiserum. The ELISA was applied to quantify lactadherin in several dairy by-products. Of the products tested, raw and commercial buttermilk had the highest concentrations of lactadherin (6.79 and 5.27 mg/g of product, respectively), followed by commercial butter serum (4.86 mg/g), commercial skim milk (4.84 mg/g), and raw whey (1.20 mg/g). The concentration of immunoreactive lactadherin was also determined in dairy by- products after they were subjected to different technological treatments. Thus, raw products were heat treated at combinations of temperature and time typically used in the dairy industry, and commercial products were hydrolyzed using 3 proteolytic enzyme preparations. Heat treatments of whey and buttermilk resulted in a smaller decrease in lactadherin concentration than did hydrolysis as determined by ELISA and electrophoresis. At high temperatures for long durations, the loss of lactadherin was higher in whey than in buttermilk, with the maximal reduction of around 48% found after treating whey at 72 degrees C for 60 min. Hydrolysis of commercial products with proteolytic enzymes resulted in a marked decrease of immunoreactivity within the first 5 min of treatment, which thereafter was constant throughout 4 h of hydrolysis. These results demonstrate that dairy by- products from milk fat processing are good natural sources of lactadherin, although technological processes have to be considered, because they have different effects on lactadherin content

Occurrence of aflatoxin contamination in maize kernels and molecular characterization of the producing organism, Aspergillus

Aflatoxins are toxic metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1) is a potent carcinogen, teratogen and mutagen. 660 pre- and post- harvest maize samples were collected from major maize growing areas in Tamil Nadu, India. Aflatoxin contamination was observed in 40.22% of the samples tested of which, 22.97% of pre-harvest and 53.93% post-harvest maize samples were found to be infected with AFB1 and 12.05% of the total samples exceeded WHO permissible limit of 20 μg/kg. AFB1 contamination ranged from 0 to 149.32 μg/kg. 28 A. flavus isolates were isolated and grouped into three sets based on aflatoxin producing potential viz., highly aflatoxin producing isolates, medium producing isolates and no aflatoxin producer or traces of aflatoxin producing isolates. The genetic coefficient matrix analysis using random amplified polymorphic DNA (RAPD) with ten random primers revealed minimum and maximum percent similarities among the tested A. flavus strains ranging from 35 to 89%. Cluster analysis separated the three sets of isolates into two groups (groups I and II) with each two subgroup confirming the genetic diversity among the A. flavus isolates from maize.Keywords: Maize, survey, Aspergillus flavus, aflatoxin, random amplified polymorphic DNA (RAPD).African Journal of Biotechnology Vol. 12(40), pp. 5839-584

Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol (DON)

Deoxynivalenol (DON), one of the trichothecene mycotoxins, is a worldwide contaminant of wheat and barley, especially when infected by Fusarium graminearum, the causative agent of an epidemic wheat disease called Fusarium Head Blight. Because of the high risk of DON ingestion and the possibility of frequent exposure, it is important to develop a rapid and highly sensitive method for easy identification and quantification of DON in grain samples. In this study, we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect DON in wheat. We conjugated 3-O-Hemisuccinyl-DON (3HS-DON) to Bovine serum albumin (BSA) and Ovalbumin (OVA), and obtained DON-specific mice antisera. The indirect competitive ELISA revealed that the optimal concentration of mice serum and the coated antigen was 1/1600 and 1/1500, respectively. The antiserum cross-reacted with the trichothecenes 3-acetyl-DON and T-2 toxin, reaching about 55.2% and 6.3%, respectively, as compared with DON. Results showed that the assay could be performed satisfactorily using an extraction buffer containing less than 15% methanol. Recovery from DON was 82–93% in grains. The linear detection range of DON in grains was between 0.01 and 100 μg/mL